Drugs for prevention and treatment of diseases caused by abnormalities in cartilage tissues

ABSTRACT

Drug or foods and drink for prevention or treatment of diseases caused by abnormalities in cartilage tissues in which said drugs or foods and drinks have omega-9 unsaturated fatty acid as an active ingredient.

TECHNICAL FIELD

[0001] The present invention relates to drugs for prevention ortreatment of diseases caused by abnormalities in cartilage tissues,containing an omega-9 unsaturated fatty acid as an active ingredient,and foods and drinks having the effect of preventing or alleviatingmedical conditions caused by abnormalities in cartilage tissues, and toa process for producing said drugs. More specifically, the presentinvention relates to drugs for prevention or treatment of diseasesassociated with cartilage degeneration such as arthropathy,osteoarthritis, gonarthrosis, periarthritis scapulo-humeralis,spondylitis deformans, etc. and foods or drinks having the effect ofpreventing or alleviating joint pains, cinealgia, limited range ofmotion of joints, swelling joints or hydrarthrosis, etc. containing asan active ingredient at least one compound selected from the groupconsisting of 6,9-octadecadienoic acid, 8,11-eicosadienoic acid, and5,8,11-eicosatrienoic acid, and to a process for producing them.

BACKGROUND ART

[0002] Arthropathy has been divided into the secondary arthropathy thatoccurs as a sequela to some underlying disease that causes cartilagedegeneration, and the primary arthropathy having no specific underlyingdiseases. Arthropathy is seen most frequently in the knee joints, andless frequently in the joints of elbow, hip, legs, and fingers. A changeto a morbid state causes mainly degeneration of cartilage and therepairing mechanism thereof. The progress of the morbid state leads tochanges in synovial membrane, destruction of subchondral bones,cartilage hypertrophy in the joint margins, or neogenesis of bone.

[0003] Osteoarthritis is a disease in which chronic degenerative changesand proliferative changes take place simultaneously in joints leading tochanges in morphology of joints, and is roughly classified into theprimary osteoarthritis and the secondary osteoarthritis. The former ismore frequently seen in and after middle ages, wherein aging processescombined with mechanical stress lead to its onset, and the latter isseen in the young generation as well, with its onset being secondary tosome evident etiology such as joint trauma, morphological abnormalities,diseases, metabolic disorders, etc. Histologically it is characterizedby cartilage abrasion as a result of biochemical changes of cartilage,with cleavage as well as reduced viscoelasticity of cartilage reachingthe deep part. Spondylosis deformans is a degenerative lesion of thespine. Although its main cause is age change, involvement ofconstitutional disposition, environmental factors such as occupation,trauma in a broader sense, etc. has been implicated (cited from “MedicalDictionary, published on Mar. 25, 1991, Nannzanndo”).

[0004] Especially, with regard to the primary osteoarthritis, theincreasing number of patients due to underlying causes of aging of thepopulation has become a major social problem. As a medicinal treatmentof osteoarthritis, administration of nonsteroidal anti-inflammatorydrugs, intra articular injection of steroids, etc. have been employed inorder to prevent inflammation of articular cartilage and periosteum, butthese are all symptomatic treatments and, therefore, have not been verysuccessful at present for arresting the progress of cartilagedestruction.

[0005] As a protection against extraneous cartilage destruction due tomechanical stress etc., a high-molecular hyaluronic acid preparation hasbeen used, but it cannot be considered an essential method of treatment.Ameliorating drugs of endogeneous cartilage destruction due to metabolicdisorders of chondrocytes themselves which are becoming evident in therecent progress of biochemical research on chondrocytes, and drugs forcausative treatment such as would regenerate cartilage loss ordestruction often occurring in osteoarthritis have not been known, andits development has been strongly desired.

DISCLOSURE OF THE INVENTION

[0006] Thus, it is an object of the invention to provide drugs that areuseful for prevention or treatment of diseases caused by abnormalitiesin cartilage tissues, in particular, for prevention of diseaseassociated with cartilage degeneration such as, in particular,arthropathy, osteoarthritis, periarthritis scapulo-humeralis,spondylitis deformans, etc. and that have relatively small side effects,and thus are applicable to chronic disorders, and new foods and drinkshaving the effect of preventing or alleviating medical conditions causedby abnormalities in cartilage tissues.

[0007] Cartilage tissues are avascular, which is extremely uncommon as atissue derived from a mesenchyma, and even normal cartilage tissuescontain lipids (Ann. Rheum. Dis., 24, 123-135, 1965). However, excessiveaccumulation of lipids may be observed as pathomorphism in lipo-hemarthropathy (J. Bone Joint Surg. [Am] 52, 1147-1158, 1970) andprecocious arthropathy (Arthritis Rheum. 24, 965-968, 1981). Inaddition, correlation of abnormal metabolism of lipids with medicalconditions has been reported in osteoarthritis as well (Metabolism, 40,571-576, 1991).

[0008] An appropriate amount of lipids and appropriate composition offatty acids are considered to be important for maintenance of normalmetabolism and functions of chondrocytes based on the reports thatadministration of lipids into a joint of rabbit can induce experimentalosteoarthritis-like arthropathy (J. Anat, 133, 309-314, 1981), and canreduce chondral incorporation of ³⁵SO₄ in essential fattyacids-deficient Wistar rats (Arthritis Rheum., 14, 379, 1971), and thatlipid supply is needed for normal growth of chondrocytes (Exp. Cell.Res., 145, 415-423, 1983).

[0009] However, the role of the above changes in lipids in initiationand aggravation processes of arthropathy has not been fully studied, andfurthermore it is not clear whether the administration of lipids iseffective for treatment of arthropathy, especially, osteoarthritis. Onthe other hand, omega-9 unsaturated fatty acids are known to belocalized in cartilage tissues and its contents decrease with aging(FASEB J., 5,344-353, 1991), and believed to be important formaintenance of functions of chondrocytes. Furthermore,5,8,11-eicosatrienoic acid has been confirmed to have ananti-inflammatory effect on the rat models of carrageenan-induced edema,but its pharmaceutical actions in arthropathy is little known.

[0010] Thus, in order to resolve the above problems, the inventors havecarried out studies on various unsaturated fatty acids and consequentlyfound that 5,8,11-eicosatrienoic acid has an action of inhibitingcartilage degeneration and that hence it is very useful for preventionor treatment of diseases caused by abnormalities in cartilage tissues,and we have completed the present invention.

BRIEF EXPLANATION OF THE DRAWINGS

[0011]FIG. 1 is a graph showing a therapeutic effect of mead acid on arabbit model of osteoarthritis.

[0012]FIG. 2 is a graph showing a mead acid/arachidonic acid ratio inthe articular cartilage tissue of a rabbit model of osteoarthritis.

[0013]FIG. 3 is an electrophoretic pattern showing the effect of variousfatty acids on the synthesis of type II collagen by a chondrocyte

MODE FOR CARRYING OUT THE INVENTION

[0014] Omega-9 unsaturated fatty acid, an active ingredient of thepresent invention, is 5,8,11-eicosatrienoic acid, and6,9-octadecadienoic acid or 8,11-eicosadienoic acid which can be readilyconverted to 5,8,11-eicosatrienoic acid thereby showing the effect of5,8,11-eicosatrienoic acid may be employed. They may be used alone or incombination.

[0015] The fatty acids of the present invention may be any of thegeometric isomers, the cis-form or the trans-form. But since most of thenaturally occurring omega-9 unsaturated fatty acids are in the cis-form,the omega-9 unsaturated fatty acids are preferably in the cis-form whenused in foods and drinks of the present invention.

[0016] Omega-9 unsaturated fatty acids according to the presentinvention may be employed in the form of free fatty acids, and also invarious forms, for example, pharmaceutically acceptable salts such assodium salts, potassium salts, lithium salts, or other alkali metalsalts, salts of other metals such as zinc salts, calcium salts,magnesium salts, mono-, di-, tri-glycerides, esters of lower alcohols,phospholipids, glycolipids, amides, etc. Especially ethyl esters ortriglycerides are preferred. The term “lower alcohol” as used hereinmeans monohydric alcohols having not more than six carbon atoms, such asmethanol, ethanol, propanol, isopropanol, butanol, pentanol, hexanol,etc. These may be used alone or in combination.

[0017] Sources of omega-9 unsaturated fatty acids used in the presentinvention may be any source. Thus, they may be those produced bymicroorganisms capable of producing omega-9 unsaturated fatty acids,animal tissues deficiency of essential fatty acids, and cultured cell ofanimal deficiency of essential fatty acids, chemically or enzymaticallysynthesized products, or products extracted, isolated, or purified fromnatural products for example animal cartilages.

[0018] Specific examples of microorganisms capable of producing omega-9unsaturated fatty acids refer to those microorganisms having theenzymatic activity of Δ5 desaturation and the enzymatic activity of Δ6desaturation, and having reduced or no enzymatic activity of Δ12desaturation as set forth in Japanese Unexamined Patent Publication No.5(1993)-91888. For example, Mortierella alpina SAM1861 (FERM BP-3590)may be used.

[0019] Free Omega-9 unsaturated fatty acids and their esters may beextracted, isolated, and purified from the above-mentioned omega-9unsaturated fatty acids-containing products according to theconventional method. When they are obtained from microorganisms, forexample, fats and oils obtained from a cultured cell mass by extractionwith an organic solvent such as n-hexane, or supercritical gasextraction with carbon dioxide are subjected to hydrolysis oresterification to prepare a free fatty acid mixture or a fatty acidester mixture, which is then subjected to urea fractionation,liquid-liquid partition chromatography, column chromatography, etc. toobtain the desired free fatty acids or esters of 6,9-octadecadienoicacid, 8,11-eicosadienoic acid, and 5,8,11-eicosatrienoic acid, etc. at ayield of 80% or higher. More particularly, the extraction, isolation,and purification may be carried out in accordance with the methods asset forth in Japanese Unexamined Patent Publication No. 5(1993)-91888.

[0020] The omega-9 unsaturated fatty acids, active ingredient of thepresent invention, need not be highly purified products, but fats andoils containing omega-9 unsaturated fatty acids (said fats and oils maycontain triglycerides, diglycerides, monoglycerides, phospholipids, orglycolipids having omega-9 unsaturated fatty acids, or free omega-9unsaturated fatty acids or their esters), free fatty acids mixturescontaining omega-9 unsaturated fatty acids or fatty acid ester mixturescan be used.

[0021] Oils and fats containing omega-9 unsaturated fatty acids may beobtained from a cultured cells of microorganism capable of producingomega-9 unsaturated fatty acids by disrupting the cells and byextraction with an organic solvent such as n-hexane, or supercriticalgas extraction with carbon dioxide. Alternatively, they may be obtainedfrom a natural product containing omega-9 unsaturated fatty acids by theconventional method. Hydrolysis and esterification of the thus obtainedoils and fats can yield a free fatty acid mixture or fatty acid estermixtures containing omega-9 unsaturated fatty acids. More particularly,the extraction, isolation, and purification from the cultured cells maybe carried out in accordance with the method as set forth in JapaneseUnexamined Patent Publication No. 5(1993)-91888.

[0022] The fatty acids of the present invention may be used orally orparenterally in the field of drugs, quasi-drugs, cosmetics,health-related foods, functional foods, nutritional supplementary foods,foods for the elderly, and foods and drinks.

[0023] Diseases caused by abnormalities in cartilage tissues, thesubject of the present invention, include, for example, arthropathy ofthe joints of knees, elbows, hip, legs, fingers, shoulders, etc.,osteoarthritis, gonarthrosis, periarthritis scapulo-humeralis,spondylitis deformans, etc. Various medical conditions caused by thesediseases are also the subject of the present invention, and includestiffness of joints, cinealgia, limited range of motion of joints, andswelling of joints known as disease conditions of osteoarthritis, pains,especially, cinealgia, limited range of motion, and hydrarthrosis knownas disease conditions of gonarthrosis, trachelodynia, back pain,lumbago, irradiating pains of limbs or sometimes the trunk,radiculopathy or myelopathy such as numbness or weakness, highparalysis, bladder and rectal disturbances, intermittent claudication,or cold sensation known as disease conditions of spondylitis deformans.The fatty acids of the present invention have relatively small sideeffects due to selective inhibition of cartilage degeneration, thuscapable of being applied to chronic conditions.

[0024] When the active ingredients of the present invention are employedas pharmaceutical drugs, they may be administered in any formconveniently employed for oral or parenteral administration, such asinjections (emulsifiable, suspendable, non-aqueous, etc.), or solidinjections emulsified or suspended prior to use, transfusion solutions,powders, granules, tablets, capsules, enteric coated tablets, troches,liquid for internal use, suspensions, emulsions, syrups, liquids forexternal use, fomentations, nasal drops, inhalants, ointments, lotions,suppositories, enteral nutrients, etc. They may be used either alone orin combinations depending on the disease conditions. These may beprepared according to the conventional methods by adding to the maindrug pharmacologically and pharmaceutically acceptable adjuvants formanufacture.

[0025] Such adjuvants for manufacture used are suitable components formanufacture selected depending on the route of administration such aspreparations for internal use (oral drugs), parenteral preparations(injections), drugs for application to mucosa (buccals, troches,suppositories, etc.), preparations for external use (ointments, patches,etc.), and the like. In the oral drugs and drugs for application tomucosa, for example, components for pharmaceutical manufacture such asexcipients (for example, starch, lactose, crystalline cellulose, calciumlactate, magnesium metasilicate aluminate, and anhydrous silicic acid),disintegrators (for example, carboxy methyl cellulose, calcium carboxymethyl cellulose, and sodium alginate), lubricants (for example,magnesium stearate, paraffin sulphate, and talc), binders (for example,dimethyl cellulose, gelatin, and polyvinylpyrrolidone), coating agents(for example, hydroxyethylcellulose), corrigents, coloring agents,flavoring agents, etc. may be used. In the injections, components forpharmaceutical manufacture such as solubilizing agents and solutionadjuvants (for example, distilled water for injection, physiologicalsaline, propylene glycol, and glycerin), capable of forming aqueousinjections, suspending agents (for example, surfactants such aspolysorbate 80, tween 80, etc., and gum arabic solution), pH-adjustingagents (for example, organic acids or their metal salts), stabilizers,etc. may be used. In addition, in the drugs for external use componentsfor pharmaceutical manufacture such as aqueous or non-aqueoussolubilizing agents or solution adjuvants (for example, alcohols, andfatty acid esters), adhesives (for example, carboxy vinyl polymers, andpolysaccharides), emulsifying agents (for example, surfactants), and thelike may be used. Since the fatty acids of the present invention areunsaturated fatty acids, it is preferred that they contain anti-oxidantssuch as butyrated hydroxy toluene, butyrated hydroxy anisole, propylgallate, pharmaceutically acceptable quinones, α-tocopherols, etc.Especially for treatment of arthropathy, osteoarthritis, gonarthrosis,periarthritis scapulo-humeralis, etc., an active ingredient of thepresent invention may be suspended in an aqueous solvent, its pH isadjusted to be 6.0 to 7.0 and its osmotic pressure ratio vs. 0.9%physiological saline is adjusted to 0.8 to 1.2 to prepare a dosage formfor articular cavity administration. As the above aqueous solvent, forexample, physiological saline, a 3 to 5% glucose solution, a 3 to 5%xylitol solution, phosphate buffer, etc. may be used.

[0026] As a solubilizing agent for preparing injections, for example,nonionic surfactants for pharmaceutical use, etc. may be used. Morespecifically, it may be prepared by completely mixing an activeingredient of the present invention with half volume of a nonionicsurfactant such as POE (60) hydrogenated castor oil or POE sorbitanmonooleate, etc. and then by diluting with physiological saline. Asneeded, isotonicity, oxidation inhibitor (sodium sulfite, sodiumbisulfite, etc.), preservatives (benzoic acids, salicylic acid, etc.),soothing agents, etc. may be optionally added.

[0027] In the drugs for prevention or treatment of the presentinvention, active ingredients of the present invention may be blendedwith drugs such as hyaluronic acid etc. traditionally used for treatmentof diseases caused by abnormalities in cartilage tissues,adrenocorticotropic hormone drugs, local anesthetics, antibiotics, etc.may be added.

[0028] Though the dose of the active ingredient of the present inventionmay vary depending on the purpose of administration and the status (sex,age, weight, etc.) of the patient to be administered, the total amountof the omega-9 unsaturated fatty acids of the present invention, whenorally administered to an adult, is in the range of 1 to 5,000 mg perday, preferably 1 to 2,000 mg per day, and more preferably 1 to 1,000 mgper day, and when administered parenterally it is in the range of 0.1 to500 mg per day, preferably 0.1 to 200 mg per day, and more preferably0.1 to 100 mg per day. The dose may be controlled as appropriate withinthe above-mentioned range.

[0029] For treatment of arthropathy, osteoarthritis, gonarthrosis,periarthritis scapulo-humeralis, in particular, it is injected intoarticular cavity for an adult in amounts in the range of 0.1 to 500 mgper each administration, preferably 0.1 to 200 mg per eachadministration, which is administered once every 1 to 7 days. However,in contrast to the commercially available hyaluronic acid preparation ofwhich administration is limited to injection into the articular cavitydue to its property as a protecting agent against mechanical stress, themethod of administration of the present invention is not limited toinjection into the articular cavity but it may be administered invarious methods such as oral administration, enteral nutrients,percutaneous absorption, etc., because the fatty acids of the presentinvention has the activity of inhibiting degeneration of cartilagetissues.

[0030] Fatty acids, active ingredients of the present. invention, areknown to be synthesized in the body at deficiency state of essentialfatty acids. It is evidently superior in safety because its continuouslyorally administration in an amount of 2 g/day/kg to 7-week old ICR malemice for two weeks did not cause any abnormal conditions.

[0031] When the fatty acids of the present invention are used in foodsand drinks, they may be in the form of solid or liquid foods or favoritefoods.

[0032] The foods containing oils and fats include natural foodscontaining oils and fats such as meat, fish, nuts, etc., foods to whichare added oils and fats on cooking such as chinese foods, ramen noodles,soup, etc., foods for which oils and fats are used as a heating mediumsuch as tenpura (deep-fried fish and vegetables), fried foods, friedbeancurd, fried rice, doughnuts, fried dough cake, etc., fat and oilfoods or processed foods for which oils and fats are added duringprocessing such as butter, margarine, mayonnaise, dressing, chocolate,instant ramen noodles, caramel, biscuits, cookies, cake, ice cream,etc., foods on which oils and fats are sprayed or applied at the finishof processing such as okaki (rice crackers), hard biscuits, anpan(bean-jam buns), etc., but they are not limited to the above, andinclude, for example, agricultural products such as bread, noodles,rice, confectionery (candy, chewing gum, goumis, tablet candies, andJapanese cake), bean curd and other processed foods thereof etc.,fermentation products such as refined sake, medicinal drinks, mirin(sweet sake), cooking vinegar, soy sauce, miso (fermented soy beanpaste), etc., dairy products such as yogurt, ham, beacon, sausage, etc.,processed marine products such as kamaboko (boiled fish paste), ageten(deep-fried patty of fish paste), hanpen (light, puffy cake made ofground fish), etc., beverages such as fruit juice drinks, soft drinks,sports drinks, alcohol beverages, tea, etc.

[0033] Foods and drinks of the present invention are prepared by mixinga given amount of at least one omega-9 unsaturated fatty acid selectedfrom the group consisting of 6,9-octadecadienoic acid,8,11-eicosadienoic acid, and 5,8,11-eicosatrienoic acid, or extractscontaining one of them as a main ingredient, with a source for food anddrink containing substantially no omega-9 unsaturated fatty acids, andthen by processing it according to the conventional method ofmanufacture. The amount mixed may vary depending on the forms andproperty of the food and it is generally 0.001% by weight or more of thetotal amount of the food, preferably 0.1% by weight or more, and morepreferably 1% by weight or more, but is not limited to the above.

[0034] The sources for food and drink containing substantially noomega-9 unsaturated fatty acids according to the present inventioninclude those in which the total amount of 6,9-octadecadienoic acid,8,11-eicosadienoic acid, and 5,8,11-eicosatrienoic acid is less than onemg, and preferably less than two mg, due to the small amount of omega-9unsaturated fatty acids contained, or those in which the content of5,8,11-eicosatrienoic acid is less than one mg, and preferably less thantwo mg as a daily dose of the final product to which the source for foodor drink has been added.

[0035] In accordance with the present invention, the extracts containingan omega-9 unsaturated fatty acid as a main ingredient include the fatsand oils (said fats and pils may contain triglycerides, diglycerides,monoglycerides, phospholipids, and glycolipids having omega-9unsaturated fatty acids, or free omega-9 unsaturated fatty acids ortheir esters, etc.) containing omega-9 unsaturated fatty acids obtainedfrom the cultured cells of a microorganism capable of producing omega-9unsaturated fatty acids by disrupting the cells through extraction withan organic solvent such as n-hexane or through supercritical gasextraction with carbon dioxide, or by extraction from the naturalproducts containing omega-9 unsaturated fatty acids by the conventionalmethods, mixtures of free fatty acids or mixtures of fatty acid esterscontaining omega-9 unsaturated fatty acids. The content of the omega-9unsaturated fatty acids in said extracts is 1% or higher, preferably 5%or higher, and more preferably 10% or higher, and in particular, thecontent of 5,8,11-eicosatrienoic acids is 1% or higher, preferably 5% orhigher, and more preferably 10% or higher.

[0036] When used as health-related foods, functional foods, nutritionalsupplementary foods, and foods for the elderly, their forms may be theabove pharmaceutical drug form, or, for example, the processing formssuch as natural liquid diets, semi-digested nutritious foods, andchemical defined diets incorporating, in addition to the present fattyacids, for example, proteins (as the protein source, highly nutritiousmilk protein having balanced amino acid compositions, bean protein, andegg albumin protein are most widely used, but the digests thereof,oligopeptides of egg whites, hydrolyzates of beans, etc. and singleamino acids may be used), saccharides, fats, trace metals, vitamins,emulsifying agents, flavors, etc., health-related drinks, capsules,enteral nutrients, etc., but the form of the above foods and drinks arealso acceptable.

[0037] The health-related foods, functional foods, nutritionalsupplementary foods, and foods for the elderly can be produced in theform of powders, granules, tablets, capsules, troches, liquid forinternal use, suspensions, emulsions, syrups, health-related drinks,natural liquid diets, semi-digested nutritious diets, chemical defineddiets, enteral nutrients, etc. using omega-9 unsaturated fatty acidsand/or the extract containing omega-9 unsaturated fatty acids as a mainingredient. More specifically, they may be produced in the form ofpowders, granules, tablets, capsules, troches, liquid for internal use,suspensions, emulsions, syrups, health-related drinks, natural liquiddiets, semi-digested nutritious diets, chemical defined diets, enteralnutrients, etc., using oil or fat containing at least 1%, preferably atleast 5%, more preferably at least 10% of omega-9-9 unsaturated fattyacids. At this time, any of the elemental diets or any of functionalingredients may be mixed with oils and fats containing omega-9unsaturated fatty acid of the present invention.

[0038] Furthermore, under the supervision of a nutrician based ondirections by the physician, the meal prepared on site by adding thefatty acids of the present invention to any foods at the time of cookingof hospital diets can be given to patients with medical conditionscaused by abnormalities in cartilage tissues.

[0039] The medical conditions according to the present invention causedby abnormalities in cartilage tissues include various medical conditionscaused by diseases such as arthropathy of the joints of knees, elbows,hip, legs, fingers, shoulders etc. osteoarthritis, gonarthrosis,periarthritis scapulo-humeralis, spondylitis deformans, etc., and morespecifically the medical conditions including stiffness of joints,cinealgia, limited range of motion of joints, and swelling of jointsknown as disease conditions of osteoarthritis, pains, especially,cinealgia, limited range of motion, and hydrarthrosis known as diseaseconditions of gonarthrosis, trachelodynia, back pain, lumbago,irradiating pains of limbs or sometimes the trunk, radiculopathy ormyelopathy such as numbness or weakness, high paralysis, bladder andrectal disturbances, intermittent claudication, or cold sensation knownas disease conditions of spondylitis deformans.

[0040] The foods and drinks containing the fatty acids of the presentinvention are preferably administered orally, for the purpose ofpreventing or alleviating medical conditions caused by abnormalities incartilage tissues and maintaining health, in amounts of 1 to 5,000 mgper day of the total amount of the omega-9 unsaturated fatty acids ofthe present invention, preferably 1 to 2,000 mg per day, and morepreferably 1 to 1,000 mg per day.

EXAMPLES

[0041] The present invention will now be explained more particularlywith reference to the following examples.

Example 1 A Method of Preparing 5,8,11-cis-eicosatrienoic Acid EthylEster Using a Microorganism Having the Ability of Producing an omega-9Unsaturated Fatty Acid

[0042] As the microorganism having the ability of producing an omega-9unsaturated fatty acid, Mortierella alpina SAM1861 (FERM BP-3590), asset forth in Japanese Unexamined Patent Publication No. 5(1993)-91888,having the enzymatic activity of Δ5 desaturation or the enzymaticactivity of Δ6 desaturation and having no enzymatic activity of Δ12desaturation was used. The medium (7 tons, pH 6.3) containing 2%glucose, 1% yeast extract, and 0.1% olive oil was fed into a 10-ton tankand was sterilized, and then the aerated shaking culture of Mortierellaalpina SAM1861 was carried out for 12 days under the condition of atemperature of 24° C. (from day 0 to day 10 of culture) and 20° C. (fromday 10 to day 12 of culture), with aeration at 1.0 vvm, and an agitationat 50 rpm. Feeding culture of glucose was carried out so that the totalamount of glucose added was 3.49%. After the culture, granulating dryingwas conducted to obtain 6.8 kg of the dried cells of the microorganismcontaining 32.7% of the oils and fats containing omega-9 unsaturatedfatty acids. The oils and fats contained in the dried mass obtained wereextracted with hexane, purified in the conventional method to obtain 20kg of oils and fats containing omega-9 unsaturated fatty acids (13.2% of6,9-cis-octadecadienoic acid, 3.5% of 8,11-cis-eicosadienoic acid, and11.2% of 5,8,11-cis-eicosatrienoic acid). And then the oils and fatsobtained were subjected to esterification, vacuum distillation, and highperformance liquid chromatography purification to obtain 260 g of5,8,11-cis-eicosatrienoic acid ethyl ester (90.1% of5,8,11-cis-eicosatrienoic acid, 7.9% of 6,9-cis-octadecadienoic acid,1.4% of oleic acid, and 0.6% of arachidic acid).

Example 2

[0043] Models of osteoarthritis were prepared using arthrodesis by cast.The animals used were 12-week old male rabbits (Kbl: NZW, KitayamaLabesu K. K.). Under anesthesia by intramuscular administration of 2mg/kg of xylazine hydrochloride (Selactal, Bayer A. G.) and 50 mg/kg ofketamine hydrochloride (Ketalar 50 for animals, Sankyo K.K.)(hereinafter referred to as “under anesthesia”), hair was cut off theright hind leg. Then after the region from the upper thigh to the toewas covered with the stockinette and cast pad (3M Medicine K.K.), theknee joint was fixed for three weeks at a stretched and bent position bycasting tape (3M Medicine K.K.).

[0044] The animals that did not show any abnormality at the completionof arthrodesis by cast were selected and divided into four groupsconsisting of 4 to 5 animals per group by weight and range of motion ofjoints so that the average weight and the range of motion of joints ofeach group became uniform.

[0045] Samples administered were prepared by completely mixing 90% of5,8,11-cis-eicosatrienoic acid ethyl ester (hereinafter referred to as“mead acid”) obtained in Example 1 and 62.5 μl of HCO-60 (POE(60)hydrogenated castor oil, Nikko Chemical K.K.), which was then suspendedin 100 ml of physiological saline to prepare a 1 mg/ml solution of meadacid. Separately 62.5 μl of HCO-60 was dissolved in 100 ml ofphysiological saline and was used for preparation of low-dose samplesand for the solvent administration group.

[0046] The administration groups were established as follows: (1)solvent administration group: 0.6 ml/joint, (2) mead acid low-doseadministration group: 0.12 mg mead acid/joint, (3) mead acid high-doseadministration group: 0.60 mg mead acid/joint, (4) Artz (registeredtrade mark) (Seikagaku Kogyo K.K.): 0.6 ml (contains 6 mg ofhigh-molecular sodium hyaluronate)/joint. After cutting hair off theadministration site under anesthesia and disinfecting the site with 2%Isodine (Isodine for animals, Meiji Seika K.K.) and 70% ethanol,administration was carried out using a disposable syringe (injectionneedle: 25 G). Dosage samples were administered in the cavity of rightknee joint twice per week for five weeks. As an evaluation ofpharmaceutical efficacy, determination of the range of motion was madeonce a week by applying a load of about 400 g at right angle to thetibial axis and measuring an angle formed against the femoral bone. Thedifference (maximum stretching angle)-(maximum bending angle) was madethe range of motion (ROM) of the right knee joint.

[0047] During the experiment, the animals were given free access to thesolid feed RC4 (Oriental Kobo K.K.) and public tap water as the drinkingwater.

[0048] After the experiment is over, the pathological examination of thejoint was carried out. Thus, under anesthesia with administration ofsodium pentobarbital solution (Nembutal injection, Dainabott K.K.) intothe auricular vein, observation of the outer surface of the knee jointand ROM measurement were carried out and then the animals were bled todeath. After the both hind legs of all the animals were removed, theircavity of right knee joint were visually observed and photographed. Forall the animals the femoral bone, tibia epiphysial, and synovialmembrane were collected, fixed in 10% neutral buffered formalin solutionand then demineralization with EDTA was carried out. After embedding andslicing, the section was subjected to hematoxylin-eosin (H-E) stain andsafranine O stain and then observed under microscope.

[0049] ROM of the joint of the right knee is shown in FIG. 1. One weekafter the start of administration, mead acid high-dose group and theArtz (registered trade mark) administration group have displayedsignificant increase in ROM, and by week five it returned to 87% of thepre-constraint value. In the low-dose mead acid group the tendencytoward recovery was observed from week two after administration. Resultsof analysis of fatty acid composition of cartilage tissue at the time ofcompletion of administration are shown in FIG. 2. As compared with thearachidonic acid/mead acid ratio (ARA/MA) of articular cartilage tissueof the untreated rabbits, it became apparent the solvent administrationgroup tended to show elevated values, and the mead acid administrationgroup to return to normal.

Example 3

[0050] After 125 μl of 95% 6,9-cis-octadecadienoic acid ethyl ester or125 μl of 95% 8,11-cis-eicosadienoic acid ethyl ester prepared in thesame manner as in Example 1 were completely mixed with 62.5 μl of HCO-60(POE(60) castor oil, Nikko Chemical K.K.), 1 mg/ml solution of6,9-cis-octadecadienoic acid ethyl ester (solution A) or 1 mg/mlsolution of 8,11-cis-eicosadienoic acid ethyl ester (solution B)suspended 100 ml of physiological saline were prepared. Separately 62.5μl of HCO-60 was dissolved in 100 ml of physiological saline and wasused as the solvent administration group.

[0051] In accordance with Example 2, models of osteoarthritis of theknee were prepared using arthrodesis by cast and were divided into threegroups of five animals per group so that the average weight and therange of motion of joints of each group became uniform. Theadministration groups were established as follows: (1) solventadministration group: 0.6 ml/joint, (2) 6,9-cis-octadecadienoic acidethyl ester administration group: 0.6 ml solution A/joint, (3)8,11-cis-eicosadienoic acid ethyl ester administration group: 0.60 mlsolution B/joint. In accordance with Example 2, dosage samples wereadministered in the right articular cavity twice per week for five weeksand ROM of the joint of the right knee was measured.

[0052] As a result, ROM at week five after the start of administrationfor the solvent administration group, the solution A administrationgroup, and the solution B administration group were 70%, 80.2%, and83.6%, respectively, showing significant recovery effects.

Example 4 Preparation of Soft Capsules

[0053] Gelatin 70.0% Glycerin 22.9% Methyl paraoxybenzoate 0.15% Propylparaoxybenzoate 0.51% Water a suitable amount Total  100%

[0054] Into the soft capsule coating comprising the above ingredients,5,8,11-cis-eicosatrienoic acid ethyl ester was filled according to theconventional method to obtain soft capsules containing 180 mg percapsule.

Example 5 Preparation of Soft Capsules

[0055] The oily substance comprising 0.3% by weight of a-tocopheroladded to 99.7% of omega-9 unsaturated fatty acid-containing triglycerideoil (14.5% by weight of 6,9-cis-octadecadienoic acid, 2.3% by weight of8,11-cis-eicosadienoic acid, and 17.4% by weight of5,8,11-cis-eicosatrienoic acid) prepared from the omega-9 unsaturatedfatty acid-producing microorganism SAM1861 according to the method asset forth in Japanese Unexamined Patent Publication No. 5(1993)-91888was filled into the soft capsule coating comprising the ingredients asdescribed in Example 4, according to the conventional method to obtainsoft capsules containing 180 mg per capsule.

Example 6 Preparation of Juice

[0056] β-cyclodextrinz was dissolved in 20 ml of 20% ethanol solution inwater and, while stirring with a stirrer, 100 mg of5,8,11-cis-eicosatrienoic acid ethyl ester was added thereto followed byincubation at 50° C. for two hours. After cooling at room temperature(about one hour), the mixture was incubated at 4° C. for 10 hours whilestirring was continued. The precipitate that formed was recovered bycentrifuge, washed with n-hexane, and then lyophilized to obtain 1.8 gof an inclusion compound of 5,8,11-cis-eicosatrienoic acid ethyl ester.One gram of this powder was mixed uniformly with 10 liters of juice toprepare a juice containing 5,8,11-cis-eicosatrienoic acid ethyl ester.

Example 7 Fat Transfusion Solution

[0057] Forty grams of 5,8,11-eicosatrienoic acid ethyl ester, 360 g ofpurified soybean oil, 48 g of purified egg yolk lecithin, 20 g of oleicacid, 100 g of concentrated glycerin and 40 ml of 0.1N caustic soda wereadded and dispersed by a homogenizer, and then distilled water forinjection was added thereto to make 4 liters. This was emulsified by ahigh-pressure spray-type emulsifier to prepare a lipid emulsion. Thelipid emulsion was dispensed in 200 ml aliquots into a plastic bag andthen was subjected to high-pressure steam sterilization at 121° C. for20 min. to prepare a fat transfusion solution.

Example 8 Emulsifiable Injection

[0058] Ninety % by weight of 5,8,11-eicosatrienoic acid ethyl ester wasprepared as a parenteral emulsion in the following formulation accordingto the conventional method. The content of mead acid in the parenteralemulsion was 15% (w/v), to which was added 1.2% (w/v) of egg yolklecithin as the emulsifying agent, and its osmotic pressure was adjustedusing glycerin to be isotonic with the blood.

Example 9 Emulsifiable Injection

[0059] The omega-9 unsaturated fatty acid-containing triglyceride usedin Example 5 was prepared as a parenteral emulsion according to theconventional method. The content of the omega-9 unsaturated fattyacid-containing triglyceride in the parenteral emulsion was 10% (w/v),to which was added 1.2% (w/v) of egg yolk lecithin as the emulsifyingagent, and its osmotic pressure was adjusted using glycerin to beisotonic with the blood.

Example 10

[0060] Using the primary culture of chondrocytes of the rabbit kneejoint, the effects of mead acid on the synthesis of type II collagen wascompared with that of other fatty acids, in particular arachidonic acid.The mead acid used was 99% 5,8,11-cis-eicosatrienoic acid ethyl esterhighly purified in the same manner as in Example 1, and for arachidonicacid, oleic acid and linoleic acid, the commercially available 99% ethylesters thereof were used. Chondrocytes were prepared from the kneejoints of 4-week old Japan white rabbits in accordance with the methodof Suzuki and Shimomura [Shimomurra, Y., Yoneda, T., Suzuki, F.Osteogenesis by chondrocyte from growth cartilage of rat rib., Calcif.Tissue Int., 19:179-187, 1975]. The chondrocytes were plated at 10,000cells/well of the 96-well plate (Iwaki Glass K.K.) in (MEM medium (50μg/ml vitamin C, 1 ng/ml b-FGF were added) supplemented with 10% bovinecalf serum, and pre-incubated for two days (5% CO₂, 37° C.). The fattyacids were suspended in 0.0625% HCO-60 prior to use. As thepre-treatment with the fatty acids, the plates were divided into threegroups and added thereto the solvent (0.0625% HCO-60) (group A), meadacid (the final concentration, 300 μM) (group B), and arachidonic acid(the final concentration, 300 μM) (group C), and then incubated forabout 5 days while the culture medium and the fatty acids were replacedwith fresh ones every two days.

[0061] Subsequently, the culture medium was replaced with αMEM medium (1μg/ml transferrin, 1 μM dexamethasone, 2 μg/ml insulin, 50 μg/ml vitaminC) and each fatty acid (mead acid, arachidonic acid, oleic acid, andlinoleic acid) was added thereto to a final concentration of 300 μMfollowed by incubation for seven days to synthesize extracellularmatrix. The culture medium and each of the fatty acids were replacedevery 2 days with freshly prepared ones. Pepsin (2 mg/ml) acetatesolution was added to the plate at 200 μl/well and treated at 37° C. for24 hours. The residue was recovered from each well and was centrifugedat 15,000 rpm for 10 minutes to form a precipitate, which was thenwashed once with tris-EDTA buffer (pH 8.0) and dissolved in 100 μl ofthe SDS sample buffer.

[0062] The amount of type II collagen synthesized was determined byelectrophoresing {fraction (1/10)} volume of each sample solution on themultigel {fraction (4/20)} (Daiichi Kagaku K.K.) and measuring thedensity of the 116 kD band after staining with Cooumassie BrilliantBlue. The photograph of the stained gel is shown in FIG. 3. It revealedthat the addition of arachidonic acid (300 μM) inhibited synthesis oftype II collagen (FIG. 3, sample No. 1-5). The inhibition was moreconspicuous in result of group C pretreated with arachidonic acid butwas recovered by the addition of mead acid (300 μM) (FIG. 3, sample No.11-15). The activity of recovering the inhibited synthesis of type IIcollagen by arachidonic acid was unique to mead acid and was notobserved in other fatty acids such as oleic acid or linoleic acid.Furthermore, the result of group B pretreated with mead acid (300 μM)indicated that the pretreatment with mead acid inhibited the reductionin synthesis of type II collagen induced by the addition of arachidonicacid (FIG. 3, sample No. 6-10).

1. A drug for prevention or treatment of a disease caused byabnormalities in cartilage tissues, comprising an omega-9 unsaturatedfatty acid.
 2. A drug for prevention or treatment of a disease caused byabnormalities in cartilage tissues according to claim 1, wherein theomega-9 unsaturated fatty acid is at least one compound selected fromthe group consisting of 6,9-octadecadienoic acid, 8,11-eicosadienoicacid, and 5,8,11-eicosatrienoic acid.
 3. A drug for prevention ortreatment of a disease according to claim 1 or 2 wherein the diseasecaused by abnormalities in cartilage tissues is arthropathy.
 4. A drugfor prevention or treatment of a disease according to claim 1 or 2wherein the disease caused by abnormalities in cartilage tissues isosteoarthritis.
 5. A drug for prevention or treatment of a diseaseaccording to claim 1 or 2 wherein the disease caused by abnormalities incartilage tissues is gonarthrosis.
 6. A drug for prevention or treatmentof a disease according to claim 1 or 2 which the disease caused byabnormalities in cartilage tissues is periarthritis scapulo-humeralis.7. A drug for prevention or treatment of a disease according to claim 1or 2 wherein the disease caused by abnormalities in cartilage tissues isspondylitis deformans.
 8. Food or drink having the effect of preventingor alleviating medical conditions caused by abnormalities in cartilagetissues, comprising omega-9 unsaturated fatty acids and/or extractswhich contain an omega-9 unsaturated fatty acid as the main ingredient.9. Food or drink according to claim 8 wherein the omega-9 unsaturatedfatty acid is at least one compound selected from the group consistingof 6,9-octadecadienoic acid, 8,11-eicosadienoic acid, and5,8,11-eicosatrienoic acid.
 10. Food or drink according to claim 8 or 9wherein the food or drink is functional food.
 11. Food or drinkaccording to claim 8 or 9 having the effect of preventing or alleviatingmedical conditions caused by abnormalities in cartilage tissues, whereinthe food or drink is nutritional supplementary food.
 12. Food or drinkaccording to claim 8 or 9 having the effect of preventing or alleviatingmedical conditions caused by abnormalities in cartilage tissues, whereinthe food or drink is food for the elderly.
 13. Food or drink accordingto any of claims 8 to 12 having the effect of preventing or alleviatingmedical conditions caused by abnormalities in cartilage tissues, whereinthe food or drink is in the form of powders, granules, tablets,capsules, troches, liquid for internal use, suspensions, emulsions,syrups, health-related drinks, or enteral nutrients.
 14. A method forproducing food or drink having the effect of preventing or alleviatingmedical conditions caused by abnormalities in cartilage tissues, whereinthe omega-9 unsaturated fatty acid and/or an extract containing omega-9unsaturated fatty acid as a main ingredient is used alone or incombination with a food or drink source containing substantially noomega-9 unsaturated fatty acids.
 15. A method of preventing or treatinga disease caused by abnormalities in cartilage tissues in which theomega-9 unsaturated fatty acid is administered to a subject requiringsuch an administration.
 16. A method according to claim 15 wherein theomega-9 unsaturated fatty acid is at least one compound selected fromthe group consisting of 6,9-octadecadienoic acid, 8,11-eicosadienoicacid, and 5,8,11-eicosatrienoic acid.
 17. A method according to claim 15or 16 wherein the disease caused by abnormalities in cartilage tissuesis arthropathy.
 18. A method according to claim 15 or 16 wherein thedisease caused by abnormalities in cartilage tissues is osteoarthritis.19. A method according to claim 15 or 16 wherein the disease caused byabnormalities in cartilage tissues is gonarthrosis.
 20. A methodaccording to claim 15 or 16 wherein the disease caused by abnormalitiesin cartilage tissues is periarthritis scapulo-humeralis.
 21. A methodaccording to claim 15 or 16 wherein the disease caused by abnormalitiesin cartilage tissues is spondylitis deformans.
 22. A method according toany of claims 15 to 21 wherein the omega-9 unsaturated fatty acid isadded to a foods and drinks source.
 23. A method for producing food ordrink having the effect of preventing or alleviating medical conditionscaused by abnormalities in cartilage tissues, said foods and drinkscomprising omega-9 unsaturated fatty acids and/or extracts which containan omega-9 unsaturated fatty acid as the main ingredient.
 24. A methodaccording to claim 23 wherein the omega-9 unsaturated fatty acid is atleast one compound selected from the group consisting of6,9-octadecadienoic acid, 8,11-eicosadienoic acid, and5,8,11-eicosatrienoic acid.
 25. A method according to claim 23 or 24wherein the food or drink is functional food.
 26. A method according toclaim 23 or 24 wherein the food or drink is nutritional supplementaryfood.
 27. A method according to claim 23 or 24 wherein the food or drinkis food for the elderly.
 28. A method according to any one of claims 23to 27, wherein the food or drink is in the form of powders, granules,tablets, capsules, troches, liquid for internal use, suspensions,emulsions, syrups, health-related drinks, or enteral nutrients.
 29. Useof an omega-9 unsaturated fatty acid for producing a drug for preventionand treatment of a disease caused by abnormalities in cartilage tissues.30. The use according to claim 29 wherein the omega-9 unsaturated fattyacid is at least one compound selected from the group consisting of6,9-octadecadienoic acid, 8,11-eicosadienoic acid, and5,8,11-eicosatrienoic acid.
 31. The use according to claim 29 or 30wherein the disease caused by abnormalities in cartilage tissues isarthropathy.
 32. The use according to claim 29 or 30 wherein the diseasecaused by abnormalities in cartilage tissues is osteoarthritis.
 33. Theuse according to claim 29 or 30 wherein the disease caused byabnormalities in cartilage tissues is gonarthrosis.
 34. The useaccording to claim 29 or 30 wherein the disease caused by abnormalitiesin cartilage tissues is periarthritis scapulo-humeralis.
 35. The useaccording to claim 29 or 30 wherein the disease caused by abnormalitiesin cartilage tissues is spondylitis deformans.